Open AccessA Study of p ‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens
Author(s) -
MÜLLER Franz,
VOORDOUW Gerrit,
BERKEL Willem J. H.,
STEENNIS Pieter J.,
VISSER Servaas,
ROOIJEN Peter J.
Publication year - 1979
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1979.tb04236.x
Subject(s) - chemistry , sedimentation equilibrium , molecular mass , chromatography , dimer , enzyme , sodium dodecyl sulfate , size exclusion chromatography , sephadex , gel permeation chromatography , gel electrophoresis , tetramer , polyacrylamide gel electrophoresis , protein quaternary structure , ultracentrifuge , biochemistry , organic chemistry , protein subunit , gene , polymer
The purification procedure for p ‐hydroxybenzoate hydroxylase has been modified by replacement of the DEAE‐cellulose (DE‐32) column in the original procedure by a Sephadex‐Cibacron‐blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40–50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass ( M r ) of 43000–45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher‐order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N ‐ethylmaleimide or 5, 5′‐dithiobis(2‐nitrobenzoic acid). The iso‐electric point of the enzyme was found to be 5.8.